CN113717915B - Xanthomonas oryzae with ACC deaminase expressed on surface as well as construction method and application thereof - Google Patents
Xanthomonas oryzae with ACC deaminase expressed on surface as well as construction method and application thereof Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12Y305/99—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in other compounds (3.5.99)
- C12Y305/99007—1-Aminocyclopropane-1-carboxylate deaminase (3.5.99.7)
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- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
Abstract
The application discloses recombinant xanthomonas oryzae and a construction method and application thereof. The construction method of the recombinant xanthomonas oryzae comprises the steps of introducing encoding genes of fusion proteins into receptor xanthomonas oryzae to obtain the recombinant xanthomonas oryzae, wherein the fusion proteins are proteins formed by fusing anchor proteins used for displaying on the surface of cell outer membranes and ACC deaminase. Experiments prove that the recombinant xanthomonas oryzae constructed by the application can improve the activity of ACC deaminase on the basis of maintaining the physiological characteristics of the xanthomonas oryzae, is suitable for inoculating plants to increase the yield of the plants, and is hopeful to be further popularized and applied and developed in industry.
Description
Technical Field
The application relates to Xanthomonas oryzae with ACC deaminase expressed on the surface in the field of bioengineering, and a construction method and application thereof.
Background
The engineering strain can be used for insects, animals and plants to play the artificial design function. For example: serratia AS1, genetically engineered to function against plasmodium infection after colonisation of the anopheles' gut, and E.coli was discharged into the stool as an indicator to indicate a particular enteric pathogen infection, and antagonistic bacteria were applied to the soil or sprayed onto plants to antagonize the corresponding pathogenic bacteria.
ACC deaminase, whose coding gene is acdS, is widely found in rhizobia and various rhizosphere bacteria and fungi, and reduces ethylene levels in plants by degrading the ethylene precursor ACC (1-aminocyclopropane-1-carbonyl), thereby promoting plant growth. At present, engineering bacteria are obtained by genetic engineering of ACC deaminase, and the technology for promoting plant growth is not reported yet.
Disclosure of Invention
The application aims to solve the technical problem of how to promote plant growth by engineering bacteria.
In order to solve the technical problems, the application provides Xanthomonas oryzae with ACC deaminase expressed on the surface and a construction method thereof. The method for constructing recombinant xanthomonas oryzae provided by the application comprises the steps of introducing encoding genes of fusion proteins into receptor xanthomonas oryzae to obtain recombinant xanthomonas oryzae, wherein the fusion proteins are proteins formed by fusing anchor proteins used for displaying on the surface of cell outer membranes with ACC deaminase.
In the above method, the ACC deaminase may be a protein of X1 or X2:
the X1 and amino acid sequences are 214-551 amino acids in the sequence 2 in the sequence table;
x2, carrying out substitution and/or deletion and/or addition of more than one amino acid residue on the amino acid sequences shown in 214-551 of the sequence 2 in the sequence table to obtain a protein which has more than 80% of identity with the protein shown in X1 and has ACC deaminase activity;
x3, fusion protein obtained by fusing protein tags at the carboxyl end or/and the amino end of the protein shown in X1 or X2.
The protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag protein may be Flag tag protein, his tag protein, MBP tag protein, HA tag protein, myc tag protein, GST tag protein, SUMO tag protein, and the like.
In the above method, the coding gene of the fusion protein contains the coding gene of the ACC deaminase, and the coding gene of the ACC deaminase can be any one of DNA molecules x1-x 4:
x 1) the coding sequence (CDS) is the cDNA molecule or genome DNA of 640 th to 1656 th positions of the sequence 1 in the sequence table;
x 2) a cDNA molecule or genomic DNA which hybridizes under stringent conditions with the DNA molecule defined in x 1) and which encodes said ACC deaminase;
x 3) a cDNA molecule or genomic DNA having more than 80% identity to the DNA molecule defined by x 1) or x 2) and encoding said ACC deaminase.
In the above method, the anchoring protein may be Inak-N protein. The Inak-N protein may be a protein of Y1 or Y2:
y1 is protein composed of amino acid sequences shown in the 1 st to 211 st positions of a sequence 2 in a sequence table;
y2, and the extracellular membrane obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequences shown in the 1 st to 211 st positions of the sequence 2 in the sequence table show the protein which shows the characteristic and is derived from Y1).
In the above method, the encoding gene of the fusion protein contains the encoding gene of the anchoring protein, and the encoding gene of the anchoring protein can be any one of DNA molecules y1-y 4:
y 1) the coding sequence (CDS) is cDNA molecules or genome DNA of the 1 st to 633 st positions of the sequence 1 in the sequence table;
y 2) a cDNA molecule or genomic DNA which hybridizes under stringent conditions to the DNA molecule defined in y 1) and which encodes an anchoring protein which the cell outer membrane displays;
y 3) has more than 90% identity to the DNA molecule defined by y 1) or y 2) and encodes a cDNA molecule or genomic DNA of the cell outer membrane indicative of the expression of an ankyrin.
In the above method, the fusion protein may specifically be a protein of A1 or A2:
a1, protein composed of an amino acid sequence shown as a sequence 2 in a sequence table;
a2, protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table and has ACC deaminase activity and cell outer membrane indication display activity and is derived from A1).
In the above method, the encoding gene of the fusion protein may be any one of the DNA molecules a1 to a 3:
a1 A nucleotide sequence is cDNA molecules or genome DNA of a sequence 1 in a sequence table;
a2 Hybridizing under stringent conditions to the DNA molecule defined in a 1) and encoding a cDNA molecule or genomic DNA of the cell outer membrane indicative of the fusion protein of the displayed anchor protein with ACC deaminase;
a3 A cDNA molecule or genomic DNA having more than 90% identity to the DNA molecule defined in a 1) or a 2) and encoding a fusion protein of the displayed anchor protein with ACC deaminase is indicated by the extracellular membrane.
The stringent conditions described above may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO 4 Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO 4 Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO 4 Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO 4 Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO 4 Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
In the above method, the recombinant xanthomonas oryzae has the following characteristics: the recombinant xanthomonas oryzae expresses the ACC deaminase on the extracellular membrane surface.
In the above method, the gene encoding the fusion protein of the displayed ankyrin and ACC deaminase is introduced into the recipient bacterium via a recombinant expression vector comprising a gene expression cassette encoding the fusion protein of the displayed ankyrin and ACC deaminase. The gene expression cassette encoding the fusion protein of the displayed ankyrin and the ACC deaminase can comprise a gene encoding the fusion protein of the displayed ankyrin and the ACC deaminase and a promoter for promoting the transcription of the gene encoding the fusion protein of the displayed ankyrin and the ACC deaminase. The expression cassette may further comprise a terminator which terminates transcription of the gene encoding the fusion protein of the extracellular membrane indicative of the displayed anchor protein and ACC deaminase. Further, the gene expression cassette encoding the extracellular membrane indicating the fusion protein of the displayed dockerin protein with ACC deaminase may further comprise an enhancer sequence.
In one embodiment of the application, the recombinant expression vector containing the gene expression cassette encoding the fusion protein of the displayed anchor protein and ACC deaminase indicated by the extracellular membrane is a recombinant vector pHM1-inaK-N-acdS obtained by replacing a fragment between HindIII and EcoRI recognition sites of the pHM1 vector with a DNA fragment shown in sequence 1 of the sequence table.
In the above method, the gene encoding ACC deaminase is introduced into the recipient bacterium via a recombinant expression vector containing the gene encoding ACC deaminase.
In the method, the receptor xanthomonas oryzae is xanthomonas oryzae strain PXO99.
The recombinant xanthomonas oryzae prepared by the method is also the protection scope of the application.
In a specific embodiment of the application, the recombinant xanthomonas oryzae prepared by the method is specifically recombinant xanthomonas oryzae PXO99-pHM1-inaK-N-acdS obtained by introducing pHM1-inaK-N-acdS into the xanthomonas oryzae strain PXO99.
The application of the recombinant xanthomonas oryzae prepared by the method also belongs to the protection scope of the application. The application is the application of the recombinant xanthomonas oryzae prepared by the method or the method in any one or more of U1-U7:
u1, promoting ACC degradation;
u2, reducing ethylene content of plants;
u3, promoting plant growth;
u4, providing plant height;
u5, increasing plant root length;
u6, increasing the fresh weight of plant leaves;
u7, increasing the fresh weight of plant roots.
In the above application, the plant may be a dicotyledonous plant or a monocotyledonous plant, which may be a plant of the Gramineae family, in particular rice, such as Xanthomonas oryzae resistant rice.
Experiments prove that the recombinant bacteria constructed by the application can improve the activity of ACC deaminase on the basis of maintaining the physiological characteristics of the xanthomonas oryzae, is suitable for inoculating plants to increase the plant yield, and is expected to be further popularized and applied and developed in industry.
Drawings
FIG. 1 is an electrophoretogram of the double cleavage products of pHM1 vector and pGH-inaK-N-acdS vector. Wherein lane M:1kb maker; lane 1: pHM1 plasmid; lane 2: pHM1 after double cleavage, about 12kb in length; lane 3: pGH-inaK-N-acdS after double cleavage, wherein the length of inaK-N-acdS is about 1668bp.
FIG. 2 is a graph showing the results of measurement of ACC deaminase activity of PXO99-pHM1-inaK-N-acdS strain. The data shown in the figures are mean ± standard deviation, the number of replicates is 3, and each group of significance differences are analyzed by t-test, representing significance analysis results of P < 0.01.
FIG. 3 shows the biomass statistics of PXO99-pHM1-inaK-N-acdS or PXO99-pHM1 plants inoculated at different times. Wherein, the graph a in fig. 3 is a plant height statistical graph, the graph b in fig. 3 is a root length statistical graph, the graph c in fig. 3 is a leaf fresh weight statistical graph, and the graph d in fig. 3 is a root fresh weight statistical graph. Each dot or square represents 1 plant, and the mean and standard deviation were calculated from 3 independent inoculation experiments (3 plants each). Each group of significant differences was analyzed with t-test, representing the significant analysis result as P < 0.05, representing the significant analysis result as P <0.01, representing the significant analysis result as P <0.001, and representing the significant analysis result as P <0.0001.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof.
The experimental methods in the following examples are conventional methods unless otherwise specified.
In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The pGH vectors in the examples described below are manufactured by Shanghai JieR bioengineering Co.Ltd.
The pHM1 vector in the examples described below is described in non-patent documents "Deng, CY., zhang, H., wu, Y.et al.Proteolis of histidine kinase VgrS inhibits its autophosphorylation and promotes osmostress resistance in Xanthomonas template Nat Commun 9,4791 (2018): https:// doi.org/10.1038/s41467-018-07228-4", available to the public from the national academy of sciences microbiological study to repeat the experiments of the present application, and is not useful for other applications.
Resistant plants Xa21 to Xanthomonas oryzae (Xoo) of rice in the examples below are described in non-patent literature, "Chen X, zuo S, schwessinger B, et al an XA21-associated kinase (OsSERK 2) regulates immunity mediated by the XA and XA3 immune acceptors. Mol plant.2014; 7 (5) 874-892.Doi:10.1093/mp/ssu003", available to the public from the national academy of sciences microbiology research to repeat the experiments of the present application, are not useful for other purposes.
The preparation method of the Xanthomonas oryzae PXO99 competence in the following examples is as follows:
(1) A single colony of Xanthomonas oryzae PXO99 was picked from the PSA plate and transferred to a 500ml flask of PSA broth, 220deg.Crpm culture to OD 600 ＝0.6；
(2) Xanthomonas oryzae PXO99 was transferred to ice and left for 30min to allow the culture to cool thoroughly. Placing the centrifuge tube on ice for cooling to obtain maximum conversion efficiency, wherein the temperature of bacteria in the whole operation process is not more than 4 ℃;
(3) Transferring the Xanthomonas oryzae PXO99 into an ice-cold centrifuge tube, and centrifuging at 5000rpm at 4 ℃ for 5min to recover the Xanthomonas oryzae PXO99;
(4) Pouring out the supernatant, adding a small amount of pre-cooled 10% glycerol into the tube, re-suspending the thalli, adding more than 40ml of 10% glycerol solution, and centrifuging at 4 ℃ at 5000rpm for 5min;
(5) Pouring out the supernatant, repeating the step (4), washing at least 4 times, cleaning the ions, adding 1ml10% glycerol to resuspend the cells;
(6) The bacterial liquid is split charging, each tube is 50ul, and the bacterial liquid is immediately put into liquid nitrogen and stored at-80 ℃.
The preparation method of the PSA solid medium used in the following examples comprises the following steps: tryptone (Trytone) 10g, sucrose (Sucrose) 10g, glutamic acid (Glutamic acid) 1g, agar (Agar) 15g were dissolved in distilled water and the volume was set to 1L.
The preparation method of DF liquid culture medium used in the following examples comprises the following steps: KH (KH) 2 PO 4 4g，Na 2 HPO 4 6g，MgSO 4 ·7H 2 O0.2 g, glucose 2g, sodium gluconate 2g, citric acid 2g, (NH) 4 ) 2 SO 4 2g, 0.1mL of a first component solution and 0.1mL of a second component solution, are dissolved in distilled water, the volume is fixed to 1L, and the pH value is regulated to 7.0. The preparation method of the component one solution comprises the following steps: h 3 BO 3 10mg，MnSO 4 ·H 2 O 11.19mg，ZnSO 4 ·7H 2 O 124.6mg，CuSO 4 ·5H 2 O 78.22mg，MoO 3 10mg, dissolved in 100mL of sterilized distilled water. The preparation method of the component two solution comprises the following steps: feSO 4 ·7H 2 O100 mg is dissolved in 10mL of sterilized distilled water.
The examples described below do not contain (NH) 4 ) 2 SO 4 The preparation method of the DF liquid culture medium comprises the following steps: KH (KH) 2 PO 4 4g，Na 2 HPO 4 6g，MgSO 4 ·7H 2 0.2g of O, 2g of glucose, 2g of sodium gluconate, 2g of citric acid, 0.1mL of component one solution and 0.1mL of component two solution are dissolved in distilled water, the volume is fixed to 1L, and the pH value is regulated to 7.0. The preparation method of the component one solution comprises the following steps: h 3 BO 3 10mg，MnSO 4 ·H 2 O 11.19mg，ZnSO 4 ·7H 2 O 124.6mg，CuSO 4 ·5H 2 O 78.22mg，MoO 3 10mg, dissolved in 100mL of sterilized distilled water. The preparation method of the component two solution comprises the following steps: feSO 4 ·7H 2 O100 mg is dissolved in 10mL of sterilized distilled water.
Example 1
ACC deaminase, whose coding gene is acdS, is widely found in rhizobia and various rhizosphere bacteria and fungi, and reduces ethylene levels in plants by degrading the ethylene precursor ACC (1-aminocyclopropane-1-carbonyl), thereby promoting plant growth.
InaK-N is 211 amino acids at the N-terminus of an ice nucleoprotein (ice nucleation protein, inak) isolated from Pseudomonas syringae (Pseudomonas syringae), can be used as an anchoring motif, fused to other proteins and expressed on the extracellular membrane. The coding gene is inaK-N gene.
According to the application, the pseudomonas syringae (Pseudomonas syringae) inaK-N gene and the pseudomonas fluorescens (Pseudomonas fluorescens) acdS gene are fused, so that the target product ACC deaminase is positioned on the outer membrane of a xanthomonas oryzae (Xanthomonas oryzae pv. Oryzae, xoo) strain PXO99 through the guidance of Inak-N. The obtained genetically engineered bacteria are inoculated to a Xa21 resistant plant of rice, ACC deaminase is efficiently expressed on the cell surface through proliferation of the Xa, so that the ethylene level of the rice is reduced, and growth and development of the Xa21 plant are promoted.
The specific method comprises the following steps:
1. sequence origin
Pseudomonas syringae inaK-N gene sequences (Accession No: AF013159,5' -end 1-633 bp) and Pseudomonas fluorescens acdS gene sequences (Accession No: JQ646055, full length 1-1017 bp) were obtained in the GenBank database (https:// www.ncbi.nlm.nih.gov/GenBank /).
2. Construction of fusion protein expression vector
The fusion protein expression vector constructed in the embodiment is named pHM1-inaK-N-acdS, the vector contains a coding gene of fusion protein inaK-N+ACC deaminase named inaK-N-acdS, the nucleotide sequence of the inaK-N-acdS coding chain is shown as a sequence 1 of a sequence table (wherein, positions 1-633 are coding sequences (CDS) of inaK-N, positions 640-1656 are coding sequences (CDS) of ACC deaminase), the coded fusion protein is named inaK-N+ACC deaminase, the amino acid sequence is shown as a sequence 2 of the sequence table (wherein, positions 1-211 are inaK-N protein sequences, positions 214-551 are ACC deaminase protein sequences), and the designed anchor protein for displaying on the surface of an extracellular membrane is inaK-N of pseudomonas, and the deaminase ACC is connected in series at the N end of the pseudomonas.
The specific construction method is as follows:
2.1 construction of cloning vector pGH-inaK-N-acdS
Synthesizing a DNA molecule (the nucleotide sequence of which is the sequence 1 of a sequence table) named inaK-N-acdS, and carrying out double digestion by HindIII and EcoRI; pGH vectors were digested with HindIII and EcoRI. The two were ligated to obtain the following recombinant vector: the restriction endonuclease HindIII and EcoRI recognition site fragment (small fragment including HindIII recognition site and EcoRI recognition site) of pGH vector was replaced with inaK-N+acdS (nucleotide sequence 1 of sequence table), and the other sequences of pGH vector were kept unchanged, and the resulting recombinant vector was named pGH-inaK-N-acdS.
2.2 construction of expression vector pHM1-inaK-N-acdS
The cloning vector pHM1-inaK-N-acdS and the expression vector pHM1 were digested with restriction enzymes HindIII and EcoRI, respectively (see FIG. 1), and the inaK-N-acdS gene fragment and linearized pHM1 vector were recovered by gel electrophoresis and ligated overnight at 16℃with T4DNA ligase. The following recombinant vectors were obtained: the restriction endonuclease HindIII and EcoRI recognition site fragments (including HindIII recognition site and EcoRI recognition site fragments) of the pHM1 vector were replaced with inaK-N-acdS (nucleotide sequence 1 of the sequence table), and the other sequences of the pHM1 vector were kept unchanged, to obtain a recombinant expression vector for expressing the fusion protein Inak-N+ ACC deaminase (amino acid sequence see sequence 2), designated pHM1-inaK-N-acdS.
3. Obtaining recombinant bacteria PXO99-pHM1-inaK-N-acdS
The pHM1-inaK-N-acdS is transformed into Xanthomonas oryzae PXO99 competent, positive transformants are screened by a PSA solid medium containing 100 mug/mL spectinomycin, and a recombinant strain of Xanthomonas oryzae is obtained and named as PXO99-pHM1-inaK-N-acdS. Xanthomonas oryzae PXO99-pHM1-inaK-N-acdS is recombinant Xanthomonas oryzae obtained by introducing a gene encoding a fusion protein of Inak-N+ACC deaminase with an amino acid sequence of sequence 2 (a nucleotide sequence of sequence 1 of a sequence table) into a wild strain PXO99 of Xanthomonas oryzae.
The recombinant strain PXO99-pHM1 of Xanthomonas oryzae obtained by converting the Xanthomonas oryzae PXO99 competence with the empty vector pHM1 is used as a control.
4. Determination of ACC deaminase level expressed by engineering bacteria PXO99-pHM1-inaK-N-acdS
4.1 culturing and collecting the thalli
Selecting a PXO99-pHM1-inaK-N-acdS monoclonal strain, wherein the control is PXO99-pHM1 monoclonal strain, culturing in 20mL of PSA culture medium at 28 ℃ for 24h-48h, centrifuging at 8000rpm/min at 4 ℃ for 10min, and collecting thalli;
with 5mL of free (NH) 4 ) 2 SO 4 The cells were washed 2 times with DF liquid medium, the supernatant was removed by centrifugation after each washing, and the cells after 2 times of washing were resuspended in 7.5mL of medium free of (NH) 4 ) 2 SO 4 Is in DF liquid medium;
adding 45 mu L of 0.5mol/L ACC water solution subjected to filtration sterilization, culturing at 28 ℃ for 24 hours, inducing to generate ACC deaminase, and centrifuging at 8000rpm/min at 4 ℃ for 10min to collect thalli;
with 5mL of 0.1 mol.L -1 The cells were washed 2 times with Tris-HCl buffer (pH 7.6) and collected by centrifugation for further enzyme activity assay.
4.2 enzyme Activity assay
The PXO99-pHM1-inaK-N-acdS or PXO99-pHM1 strain obtained in 4.1 above was resuspended in 600. Mu.L of 0.1 mol.L, respectively -1 Tris-HClBuffer (pH 8.5);
two different sets of treatments were performed on each of the 600 μl suspensions, one treatment being: adding 30 mu L of toluene into 600 mu L of bacterial suspension, and rapidly shaking for 30s to break cells; the second treatment is as follows: toluene is not added; preparing two crude enzyme solutions from each strain;
split charging 100 μl of crude enzyme solution for determining protein concentration, leaving about 500 μl of crude enzyme solution for ACC deaminase activity determination;
the crude enzyme solution was divided into two groups of 200. Mu.L, and 20. Mu.L of 0.5 mol.L was added to one group -1 The ACC aqueous solution was mixed well and ACC was not added to group two. Placing in a water bath at 30 ℃ for reaction for 15min;
1mL of 0.56 mol.L was added -1 The reaction was terminated by HCl, centrifuged at 12000rpm/min for 5min, 1mL of the supernatant was taken, and 800. Mu.L of 0.56 mol.L was added -1 HCl and 300. Mu.L of 0.2%2, 4-dinitrophenylhydrazine solution (2 mol.L) -1 Dissolved in HCl), and preserving the temperature at 30 ℃ for 30min;
2mL of 2mol.L was added -1 NaOH was mixed uniformly, and the absorbance was measured at 540nm for 5 min.
The amount of alpha-ketobutyric acid produced by catalysis of ACC deaminase is calculated by subtracting the absorbance value of the reagent mixture without ACC from the absorbance value of the reagent and ACC.
The enzyme activity of the strain was calculated against the standard curve for alpha-ketobutyrate and the standard curve for bovine serum albumin. The expression method of ACC deaminase activity comprises the following steps: under the above reaction conditions, the unit is (mu mol of alpha-ketobutyric acid/h.mg of protein) per mg of mycoprotein per hour, which catalyzes the deamination of ACC to alpha-ketobutyric acid. The method for measuring the protein content of the crude enzyme liquid comprises the following steps: taking 100 mu L of crude enzyme solution, adding 5ml of Coomassie brilliant blue solution, mixing on a vortex mixer, developing for 3min, and measuring absorbance A at 595nm 595 The protein content of the bacteria is calculated according to a bovine serum albumin standard curve. The experiment was repeated three times.
The ACC deaminase activity of the PXO99-pHM1-inaK-N-acdS strain (see FIG. 2) was measured as 9.992. Mu. Mol. Alpha. -ketobutyric acid/h.multidot.mg protein in the toluene-free experimental group and 14.538. Mu. Mol. Alpha. -ketobutyric acid/h.multidot.mg protein in the toluene-added experimental group by the above method.
5. Phenotypic determination of engineering bacteria for promoting rice growth and development
5.1 inoculation by leaf cutting
Resistant plants Xa21 of rice against Xanthomonas oryzae (Xoo) were planted in the greenhouse, and PXO99-pHM1-inaK-N-acdS was inoculated by a leaf cutting method at 6 th week after planting to inoculate PXO99-pHM1 as a control:
two Xoo strains of PXO99-pHM1-inaK-N-acdS and PXO99-pHM1 are inoculated from-80 ℃ to PSA solid culture medium for activation, cultured overnight in PSA liquid culture medium to obtain culture solution, and the culture solution is diluted 1/100 to OD with sterile water 600nm Bacterial liquid was obtained by =0.8. And (3) dipping bacteria liquid into sterile scissors to cut the positions of 5-10cm at the tops of the rice leaves, and cutting 3 leaves per rice plant to complete a bacteria inoculation experiment.
5.2 growth-promoting phenotype assay
3 rice samples were taken for biomass detection on day 10, day 15, day 20 and day 30 after inoculation, respectively. The plant height of the overground part of the rice plant and the root length of the underground part are measured, and the weight of the overground part of the rice and the weight of the washed rice root are measured.
The results show that: PXO99-pHM1-inaK-N-acdS inoculated rice plants significantly increased the plant height (FIG. 3, panel a, 10 and 15 days post-inoculation) and root length (FIG. 3, panel b, 10 to 30 days post-inoculation) of the rice plants with PXO99-pHM1-inaK-N-acdS expressed ACC deaminase compared to control PXO99-pHM1 inoculated plants; the leaf fresh weight of the aerial parts (c-chart of fig. 3, 10 th to 30 th days after inoculation) and the root fresh weight of the underground parts (d-chart of fig. 3, 10 th to 30 th days after inoculation) are significantly increased.
The growth promoting effect of the inoculated engineering bacteria PXO99-pHM1-inaK-N-acdS rice plants is good.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
<110> institute of microorganisms at national academy of sciences
<120> Xanthomonas oryzae with ACC deaminase expressed on surface, and construction method and application thereof
<130> GNCSY212621
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1656
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atgactctcg acaaggcgtt ggtgctgcgt acctgtgcaa ataacatggc cgatcactgc 60
ggccttatat ggcccgcgtc cggcacggtg gaatccagat actggcagtc aaccaggcgg 120
catgagaatg gtctggtcgg tttactgtgg ggcgctggaa ccagcgcttt tctaagcgtg 180
catgccgatg ctcgatggat tgtctgtgaa gttgccgttg cagacatcat cagtctggaa 240
gagccgggaa tggtcaagtt tccgcgggcc gaggtggttc atgtcggcga caggatcagc 300
gcgtcacact tcatttcggc acgtcaggcc gaccctgcgt caacgtcaac gtcaacgtca 360
acgtcaacgt taacgccaat gcctacggcc atacccacgc ccatgcctgc ggtagcaagt 420
gtcacgttac cggtggccga acaggcccgt catgaagtgt tcgatgtcgc gtcggtcagc 480
gcggctgccg ccccagtaaa caccctgccg gtgacgacgc cgcagaattt gcagaccgcc 540
acttacggca gcacgttgag tggcgacaat cacagtcgtc tgattgccgg ttatggcagt 600
aacgagaccg ctggcaacca cagtgatcta attgagctca tgaacctgaa tcgttttgaa 660
cgttatccgc tgaccttcgg tccttctccc atcacgccct tgaaacgcct cagcgaacac 720
ctgggcggca aggtcgagct gtatgccaaa cgcgaagact gcaacagtgg cctggccttt 780
ggcggcaaca agacgcgcaa gctcgaatac ctgattcctg aagccatcga gcagggctgc 840
gacaccctgg tgtccatcgg cgggatccag tcgaaccaga cccgccaagt cgctgccgtc 900
gccgcccacc tgggcatgaa gtgtgtgctg gtccaggaaa actgggtgaa ctactccgac 960
gccgtgtatg accgggtcgg caatatcgaa atgtctcgca tcatgggcgc cgacgtacga 1020
ctggacgccg ccgggttcga catcggcatt cggcccagct gggagaaggc catgagcgac 1080
gtggtggagc ggggcggcaa gccgttcccg attccggcgg gttgttccga gcatccttat 1140
ggcggcctcg gcttcgtcgg tttcgccgag gaagtccggc agcaggaaca ggcactgggc 1200
ttcaaattcg actacatcgt cgtgtgctcc gtgaccggca gtacccaggc gggcatggtt 1260
gtcggtttcg ccgccgacgg ccgttcgaaa aacgtcatcg gtatcgacgc ttcggccaaa 1320
ccggaaaaga ccagggcgca gatcctgcgt atcgcccggc acaccgctga actggtggag 1380
ctgggtcgcg aaatcaccga agaggatgtg gtgctcgaca cgcgcttcgc ctatccggaa 1440
tacggtttgc cgaacgatgg cacgctggaa gccattcgtc tgtgcgggcg tcttgaaggc 1500
gtgctgactg acccggtgta tgagggcaaa tccatgcacg ggatgattga aatggtccgc 1560
cgtggcgaat tccctgaagg ctcgaaagtg ctgtacgcgc acctgggcgg ggtaccggcg 1620
ctgaatgcct acagcttctt gtttcgcaac ggctga 1656
<210> 2
<211> 551
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Thr Leu Asp Lys Ala Leu Val Leu Arg Thr Cys Ala Asn Asn Met
1 5 10 15
Ala Asp His Cys Gly Leu Ile Trp Pro Ala Ser Gly Thr Val Glu Ser
20 25 30
Arg Tyr Trp Gln Ser Thr Arg Arg His Glu Asn Gly Leu Val Gly Leu
35 40 45
Leu Trp Gly Ala Gly Thr Ser Ala Phe Leu Ser Val His Ala Asp Ala
50 55 60
Arg Trp Ile Val Cys Glu Val Ala Val Ala Asp Ile Ile Ser Leu Glu
65 70 75 80
Glu Pro Gly Met Val Lys Phe Pro Arg Ala Glu Val Val His Val Gly
85 90 95
Asp Arg Ile Ser Ala Ser His Phe Ile Ser Ala Arg Gln Ala Asp Pro
100 105 110
Ala Ser Thr Ser Thr Ser Thr Ser Thr Ser Thr Leu Thr Pro Met Pro
115 120 125
Thr Ala Ile Pro Thr Pro Met Pro Ala Val Ala Ser Val Thr Leu Pro
130 135 140
Val Ala Glu Gln Ala Arg His Glu Val Phe Asp Val Ala Ser Val Ser
145 150 155 160
Ala Ala Ala Ala Pro Val Asn Thr Leu Pro Val Thr Thr Pro Gln Asn
165 170 175
Leu Gln Thr Ala Thr Tyr Gly Ser Thr Leu Ser Gly Asp Asn His Ser
180 185 190
Arg Leu Ile Ala Gly Tyr Gly Ser Asn Glu Thr Ala Gly Asn His Ser
195 200 205
Asp Leu Ile Glu Leu Met Asn Leu Asn Arg Phe Glu Arg Tyr Pro Leu
210 215 220
Thr Phe Gly Pro Ser Pro Ile Thr Pro Leu Lys Arg Leu Ser Glu His
225 230 235 240
Leu Gly Gly Lys Val Glu Leu Tyr Ala Lys Arg Glu Asp Cys Asn Ser
245 250 255
Gly Leu Ala Phe Gly Gly Asn Lys Thr Arg Lys Leu Glu Tyr Leu Ile
260 265 270
Pro Glu Ala Ile Glu Gln Gly Cys Asp Thr Leu Val Ser Ile Gly Gly
275 280 285
Ile Gln Ser Asn Gln Thr Arg Gln Val Ala Ala Val Ala Ala His Leu
290 295 300
Gly Met Lys Cys Val Leu Val Gln Glu Asn Trp Val Asn Tyr Ser Asp
305 310 315 320
Ala Val Tyr Asp Arg Val Gly Asn Ile Glu Met Ser Arg Ile Met Gly
325 330 335
Ala Asp Val Arg Leu Asp Ala Ala Gly Phe Asp Ile Gly Ile Arg Pro
340 345 350
Ser Trp Glu Lys Ala Met Ser Asp Val Val Glu Arg Gly Gly Lys Pro
355 360 365
Phe Pro Ile Pro Ala Gly Cys Ser Glu His Pro Tyr Gly Gly Leu Gly
370 375 380
Phe Val Gly Phe Ala Glu Glu Val Arg Gln Gln Glu Gln Ala Leu Gly
385 390 395 400
Phe Lys Phe Asp Tyr Ile Val Val Cys Ser Val Thr Gly Ser Thr Gln
405 410 415
Ala Gly Met Val Val Gly Phe Ala Ala Asp Gly Arg Ser Lys Asn Val
420 425 430
Ile Gly Ile Asp Ala Ser Ala Lys Pro Glu Lys Thr Arg Ala Gln Ile
435 440 445
Leu Arg Ile Ala Arg His Thr Ala Glu Leu Val Glu Leu Gly Arg Glu
450 455 460
Ile Thr Glu Glu Asp Val Val Leu Asp Thr Arg Phe Ala Tyr Pro Glu
465 470 475 480
Tyr Gly Leu Pro Asn Asp Gly Thr Leu Glu Ala Ile Arg Leu Cys Gly
485 490 495
Arg Leu Glu Gly Val Leu Thr Asp Pro Val Tyr Glu Gly Lys Ser Met
500 505 510
His Gly Met Ile Glu Met Val Arg Arg Gly Glu Phe Pro Glu Gly Ser
515 520 525
Lys Val Leu Tyr Ala His Leu Gly Gly Val Pro Ala Leu Asn Ala Tyr
530 535 540
Ser Phe Leu Phe Arg Asn Gly
545 550
Claims (8)
1. The method for constructing the recombinant xanthomonas oryzae is characterized by comprising the following steps of: the method comprises the steps of introducing a coding gene of a fusion protein into a receptor xanthomonas oryzae to obtain recombinant xanthomonas oryzae, wherein the fusion protein is formed by fusing an anchor protein used for displaying on the surface of an extracellular membrane with ACC deaminase;
the ACC deaminase is a protein of X1 or X2:
the X1 and amino acid sequences are 214-551 amino acids in the sequence 2 in the sequence table;
x2, fusion protein obtained by fusing protein labels at the carboxyl end or/and the amino end of the protein shown in X1;
the anchor protein is a protein composed of amino acid sequences shown in the 1 st-211 st positions of a sequence 2 in a sequence table.
2. The method according to claim 1, characterized in that: the coding genes of the fusion protein comprise coding genes of ACC deaminase, and the coding genes of the ACC deaminase are cDNA molecules or genomic DNA of which the coding sequences are 640 th to 1656 th positions of sequence 1 in a sequence table.
3. The method according to claim 1, characterized in that: the encoding gene of the fusion protein comprises the encoding gene of the anchoring protein, and the encoding gene of the anchoring protein is cDNA molecules or genome DNA of which the encoding sequence is the 1 st to 633 st positions of the sequence 1 in the sequence table.
4. A method according to any one of claims 1 to 3, characterized in that: the coding gene of the fusion protein is cDNA molecules or genome DNA of which the nucleotide sequence is the sequence 1 in a sequence table.
5. The method according to claim 4, wherein: the recombinant xanthomonas oryzae expresses the ACC deaminase on the extracellular membrane surface.
6. Recombinant xanthomonas oryzae constructed by the method of any one of claims 1-5.
7. Use of the method of any one of claims 1 to 5 or the recombinant xanthomonas oryzae of claim 6 in any one or more of U1 to U7:
u1, promoting ACC degradation;
u2, reducing ethylene content of plants;
u3, promoting plant growth;
u4, providing plant height;
u5, increasing plant root length;
u6, increasing the fresh weight of plant leaves;
u7, increasing the fresh weight of plant roots.
8. The use according to claim 7, characterized in that: the plant is Gramineae plant.
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| CN103045632A (en) * | 2013-01-03 | 2013-04-17 | 吉林大学 | Protein expression plasmid and application thereof in aspect of showing protein on cell surface and automatically releasing protein |
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| CN101228277A (en) * | 2005-07-18 | 2008-07-23 | 巴斯福植物科学有限公司 | Yield increase in plants overexpressing the ACCDP genes |
| CN103045632A (en) * | 2013-01-03 | 2013-04-17 | 吉林大学 | Protein expression plasmid and application thereof in aspect of showing protein on cell surface and automatically releasing protein |
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